This proposal focuses on developing a high speed DNA sequencing system based on mass spectral analysis of Sanger dideoxy sequencing products. The current experimental goal is to sequence template DNA strands in the 1,000 base range with rates exceeding 10 megabase of raw sequence per day per machine. The sequencing method involves a mass spectroscopic (gas phase) separation and detection method. To achieve this objective, current enzymatic DNA sequencing strategies will be combined with three new technologies to produce a low-cost, easily automated sequencing method. Laser vaporization is used to transfer tagged strands of DNA into the gas phase. Once in the gas phase resonance-enhanced, multiphoton ionization is used to place precisely one unit of positive charge on each strand and then time-of-flight mass analysis is performed. The time required to obtain a single mass spectrum capable of analyzing all of the sequencing products is less than l millisecond. Such an analysis rate would accelerate the sequencing rate well in excess of 1 megabase per day. Bach of the separation and detection processes, as well as the resulting data files, are readily automated. The method is designed to replace the gel electrophoretic methods currently used to analyze the products of DNA sequencing reactions. Automation of the technology we propose would result in an extremely sensitive DNA sequencing method requiring little manual labor. The sequencing cost resulting from an instrument that uses this methodology should be less than $0.10 per sequenced base, and could potentially be less than 1 cent per base. Since we and others have already demonstrated the ability to desorb DNA longer than 500 bases in length, the majority of the work in this proposal concerns both cooling of the gas phase DNA and developing the photoionization process. To enhance the cooling we are studying new thin film systems and the integration of a pulsed nozzle into our two existing laser vaporization/laser ionization time-of-flight mass spectroscopic systems dedicated to studying DNA sequencing. To enhance the photoionization process we are studying new ionization labels for oligonucleotides, as well as investigating new ionization schemes. The ionization step has the potential of providing high mass spectral resolution (correlating to long read lengths on the template strands) and sensitivity in comparison with other mass spectral methods.